KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy

Generation of Spink5/Klk5/Klk7 mutant lines.

<p><b>(A)</b> Klk5^-/- line (depicted as Klk5^-/-Klk7^+/+Spink5^+/+), was used for preparation of Klk5^-/-Klk7^-/- mice (depicted as Klk5^-/-Klk7^-/-Spink5^+/+) by TALEN mutagenesis. Obtained Klk5^-/-Klk7^-/- mice were further crossed to a Flippase (FLPe) expressing mouse line to allow conditionally expressed Klk5, thus generating Klk7^-/- mice (depicted as Klk5^+/+(loxP)Klk7^-/-Spink5^+/+). Klk5^-/-, Klk7^-/- and Klk5^-/-Klk7^-/- lines were subsequently crossed with Spink5^+/- line to obtain Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X, i.e. double and triple KO lines. <b>(B)</b> Expression of Klk5, Klk7 and Spink5 at the mRNA level was quantified using qRT-PCR, n≥4 for each genotype, error bars represent standard deviations from mean.</p

Histological analysis of epidermis structure at E18.5 dpc and P0.

<p><b>(A)</b> Hematoxylin and eosin stained skin sections from newborn mice showed a reduced granular layer and acanthosis in epidermis of Sp5^A135X/A135X pups; no obvious defects were observed in the other groups, Scale bar, 100 μm. <b>(B)</b> Analysis of epidermal differentiation in the skin from E18.5 dpc embryos. Sections were stained with antibodies against keratin14 (Krt14), keratin6 (Krt6) and fillagrin (Flg). Increased expression of Krt14 was observed in Sp5^A135X/A135X embryos, which also strongly express the stress marker Krt6. Expression of both Krt6 and Krt14 was not altered in Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X. Flg staining revealed absence of profilaggrin granules in Sp5^A135X/A135X and Klk7^-/-Sp5^A135X/A135X embryos whereas these granules were present in wt, Klk5^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice (white arrowheads). Scale bar, 50 μm.</p

Gross phenotype of Spink5- Klk- deficient mutant lines.

<p><b>(A)</b> Phenotype of wt, Sp5^A135X/A135X, Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice 12 hours after birth. Peeling skin was observed in Sp5^A135X/A135X and Klk7^-/-Sp5^A135X/A135X and to lesser extent in Klk5^-/-Sp5^A135X/A135X pups (black arrowheads). <b>(B)</b> wt, Klk5^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at P5. Klk5^-/-Sp5^A135X/A135X pups showed dry, scaly skin while Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice had stretched and shiny epidermis, with no visual signs of dehydration. <b>(C)</b> wt and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at 3 weeks. Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice showed alopecia and growth retardation. <b>(D)</b> wt and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at 6 weeks. <b>(E)</b> Vibrissae hair obtained from P5 pups analysed by scanning electron microscopy. Klk5^-/-Klk7^-/-Sp5^A135X/A135X showed hair shaft abnormalities similar to bamboo hair; Scale bar, 30 μm. <b>(F)</b> Progression of body weight of wt, Klk5^-/-Sp5^A135X/A135X, Klk5^-/-Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/- mice, n>5, error bars represent standard errors of mean.</p

Analysis of skin-barrier abnormalities.

<p><b>(A)</b> Newborn (P0) pups were analysed by barrier penetration assay using toluidine blue (TB). Remaining barrier-defect in the area of nostrils of Klk5^-/-Klk7^-/-Sp5^A135X/A135X pups in the vicinity of nostrils is marked by black arrowhead <b>(B)</b> TEWL analysis of P0 pups as a reduction of body-weight over time, n≥4 for each genotype. Error bars represent standard errors of mean; Klk/Spink5 mutant lines were compared with the wt line using Mann-Whitney U-test at 3h and 4h, ns means “not significant“; * p < 0.05 <b>(C)</b> Epidermal barrier in P5 Klk5^-/-Sp5^A135X/A135X pups was compromised in the proximity of hair follicles <b>(D)</b> Vibrissae hair (upper panel) and dorsal skin (lower panel) obtained from P5 pups analysed using scanning electron microscopy. Defective separation of hair shafts from the root sheath in Klk5^-/-Sp5^A135X/A135X is marked by white arrowhead. Dorsal skin of Klk5^-/-Sp5^A135X/A135X mice showed complete absence of hair shafts and exposed upper parts of hair follicles (yellow arrowheads); Scale bar, 300 μm.</p

Generation of <i>Spink5</i> mutant mice.

<p><b>(A)</b> Nucleotide and amino acid sequences of human SPINK5/LEKTI (left). The two-bp deletion 398delTG in exon5 of human SPINK5 gene results in a frame-shift and PTC (red box) as described in Raghunath et al., 2004(23). Comparison of corresponding sequences in murine Spink5/LEKTI (right) where deletion of TG nucleotides at position 402 (black underline) has the same impact as in humans. <b>(B)</b> Position of critical TG nucleotides (black underline) in the exon5 of murine Spink5 gene. TALEN-binding sites are marked with red underline, position of primers for PCR screening (F1, R1, F2, R2) are denoted. As a targeting construct, a single-stranded oligonucleotide having sequences homologous to wt DNA (blue underline) flanked desired mutation. Premature STOP codon (red box) and introduced new <i>Xba</i>I recognition sequence are depicted. <b>(C)</b> RFLP analysis of targeted mice. PCR product amplified from genomic DNA using primers F1 and R1 was digested using <i>Xba</i>I enzyme. Cleavage products of 283 and 220 bp originate from the positively targeted allele, a 503 bp fragment marks the wt allele. <b>(D)</b> Expression of Spink5 mRNA was analysed by semi-qPCR analysis in Sp5^A135X/A135X mice using primers F2 and R2. Expression of GAPDH was used as a control. <b>(E)</b> Phenotype of Sp5^A135X/A135X newborn pups. Areas of peeling skin are marked with black arrowheads.</p